The invention relates to insulin metabolism and more specifically to the insulin receptor substrate (IRS-1) and the gene that encodes it.
Insulin initiates its metabolic and growth promoting effects upon binding to its tetrameric receptor (Kahn et al., 1988, J. Clin. Invest. 82:1151-1156, hereby incorporated by reference; Freychet et al., 1976, Proc. Natl. Acad. Sci. (USA), 68:1833-1837, hereby incorporated by reference; Cuatrecasas, 1972, Proc. Natl. Acad. Sci. (USA) 69:1277-1281, hereby incorporated by reference) thereby activating a kinase in the .beta.-subunit to catalyze the intramolecular autophosphorylation of specific tyrosine residues of its own .beta.-subunits (Roth, R. A., 1990, in Handbook of Experimental Pharmacology, Vol. 92, Cuatrecasas, P., and Jacobs, S., eds., Springer-Verlag, hereby incorporated by reference; Kasuga et al., 1982, Nature 298:667-669, hereby incorporated by reference; Kasuga et al., 1982, Science 215:185-189, hereby incorporated by reference). Autophosphorylation enhances receptor tyrosine kinase activity toward other protein substrates (Avruch et al., 1982, J. Biol. Chem. 256:15162-15169, hereby incorporated by reference; Roth et al., 1983, Science, 219:299-301, hereby incorporated by reference, Rosen et al., 1983, Proc. Natl. Acad. Sci. (USA) 80:3237-3240, hereby incorporated by reference). Considerable evidence demonstrates that insulin receptor tyrosine kinase activity is essential for many, if not all of the biological effects of insulin (Odawara et al., 1989, Science 245:66-68, hereby incorporated by reference; Taira et al., 1989, Science, 245:63-66, hereby incorporated by reference; Moller et al., 1988, New Engl. J. Med., 319:1526-1529, hereby incorporated by reference; Ellis et al., 1986, Cell, 45:721-732, hereby incorporated by reference; Chou et al., 1987, J. Biol. Chem. 262:1842-1846, hereby incorporated by reference; Ebina et al., 1987, Proc. Natl. Acad. Sci., (USA), 84:704-708, hereby incorporated by reference; Maegawa et al., 1988, J. Biol. Chem., 263:12629-2637, hereby incorporated by reference; Morgan et al., 1987, Proc. Natl. Acad. Sci. (USA) 84:41-45, hereby incorporated by reference). However, the exact biochemical mechanisms linking receptor kinase mediated tyrosine phosphorylation to the regulation of cellular metabolic pathways are undefined.
Tyrosine phosphorylation of several cellular proteins and enzymes has been observed during the initial cellular response to some receptor tyrosine kinase-linked polypeptide growth factors, e.g., PDGF-induced phosphorylation of phospholipase C (Meisenhelder et al., 1989, Cell 57:1109-1122, hereby incorporated by reference), 3'-phosphatidyl-inositol (PI-3) kinase (Kaplan et al., 1987 Cell 57:1021-1029, hereby incorporated by reference), and the raf-1 kinase (Morrison et al., 1989, Cell 58:649-657, hereby incorporated by reference). However, the nature of the physiologically relevant cellular protein substrates of the insulin receptor kinase has remained elusive. Although many purified proteins and synthetic peptides can be phosphorylated in vitro by isolated insulin receptors (reviewed in, Rothenberg et al., 1990a, in Handbook of Experimental Pharmacology, Vol 92, Cuatrecasas, P., Jacobs, S. eds., Springer-Verlag, hereby incorporated by reference), these reactions do not occur in vivo. When anti-phosphotyrosine antibodies are used to immunoprecipitate phosphotyrosine-containing proteins which appear in intact cultured cells during insulin stimulation, a protein of approximately M.sub.r =185 kDa, designated pp185, appears in extracts of several cell types (White et al., 1987, J. Biol. Chem. 262:9769-9777, hereby incorporated by reference; White et al., 1985, Nature 318:183-186, hereby incorporated by reference; Izumi et al., 1987, J. Biol. Chem 262:1282-1287, hereby incorporated by reference; Beguinot et al., 1988, Biochemistry 27:3222-3228, hereby incorporated by reference; Kadowaki et al., 1987, J. Biol. Chem. 262:7342-7350, hereby incorporated by reference; Condorelli et al., 1989, J. Biol. Chem. 264:12633-12638, hereby incorporated by reference). Additional phosphotyrosyl proteins of lower M.sub.r have also been described in some cell lines (Rothenberg et al., 1990a, supra; Madoff et al., 1988, Biochem. J. 252:7-15, hereby incorporated by reference; Rees-Jones et al., 1985, J. Biol. Chem. 260:4461-4467, hereby incorporated by reference; Bernier et al, 1987, Proc. Natl. Acad. Sci. (USA) 84:1844-1848, hereby incorporated by reference; Heffetz et al., 1989, J. Biol. Chem 264:10126-10132, hereby incorporated by reference; Levenson et al., 1989, J. Biol. Chem. 264:19984-19993, hereby incorporated by reference). The majority of these putative substrates are unidentified, and of all such putative insulin receptor kinase substrates no clear role in insulin signalling has yet been assigned.